CN113278609A - Regeneration method of silicon substrate nucleic acid purification column - Google Patents
Regeneration method of silicon substrate nucleic acid purification column Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
- B01D15/203—Equilibration or regeneration
Abstract
The invention discloses a regeneration method of a silicon substrate nucleic acid purification column, which comprises the steps of soaking the purification column in a solution A, slightly stirring for 10-15 hours at room temperature, washing with tap water twice, soaking the purification column in a solution B, balancing to be neutral, soaking the purification column in purified water, sterilizing for 15 minutes at 121 ℃, and drying in an oven at 60 ℃ for later use. The method can treat a large amount of purification columns at one time, the purification columns are regenerated for 5 times, and the nucleic acid adsorption rate is kept at about 95%. The method is simple to operate, has the advantages of economy, high efficiency, environmental protection and the like, and is suitable for regeneration and reutilization of the silicon substrate nucleic acid purification column commonly used in the market.
Description
Technical Field
The invention belongs to the technical field of nucleic acid purification in molecular biology, and relates to a regeneration method of a silicon substrate nucleic acid purification column for experiments, in particular to a method which is simple in operation, mild in condition and convenient for large-scale treatment and regeneration of the purification column.
Background
Nucleic acid is a carrier and transmitter of biological genetic information, is the most basic operation object of molecular biology, and the extraction and purification of nucleic acid are important components of molecular biology experiments. The traditional phenol-chloroform method for extracting and purifying nucleic acid not only has complex steps and uses harmful substances such as phenol, chloroform and the like, but also has various impurity pollutions. The centrifugal nucleic acid purifying column is usually a centrifugal structure consisting of an outer collecting pipe and an inner purifying column, and a silicon substrate membrane material filled on the purifying column can efficiently and specifically adsorb nucleic acid and remove impurity proteins and other impurities in cells to the maximum extent. In recent years, commercial silica matrix nucleic acid purification columns (centrifugal column type) can specifically adsorb nucleic acid DNA and RNA, effectively remove impurity pollution, have the characteristics of simple operation process, no use of toxic substances and the like, and are widely used for common molecular biology experiments such as genome DNA extraction, total RNA extraction, DNA purification, DNA gel recovery, plasmid DNA preparation and the like. Silica matrix nucleic acid purification columns or 96-well plates provided in numerous kits on the market are disposable and expensive, and the purification columns are directly discarded after being used, so that waste and environmental protection are not facilitated. If a laboratory uses a large number of disposable nucleic acid purification columns, the cost of the experiment is greatly increased. Further, components such as recombinant DNA and harmful nucleic acid remain in the used purification column, and if these purification columns are discarded without being treated, damage to the environment or leakage and contamination of important DNA may occur.
Currently, the recycling of such columns has received some attention from researchers. Siddappa et al (2007) soaked the nucleic acid purification column with 1M hydrochloric acid effectively removed residual DNA, but the treatment time was long. Tagliavia et al (2009), Jiangwang et al (Chinese patent 200910220752.8), Hanhan (Chinese patent 201010149814.3) and Xionghua et al (2015) are treated sequentially with alkali or acid solutionNucleic acid purification columns for treating contaminating genomic DNA are effective in removing nucleic acid residues from the columns, but these methods use high concentrations of caustic strong acids and bases (NaOH, H)2SO4HCl) and water bath condition of 65 ℃, has potential safety hazard, and the later discharge of the treatment fluid is not beneficial to ecological environment protection. Liu Jie Qing et al (2014) etc. used DNA purification columns are placed in 0.01mol/L phosphate buffer solution with pH 2.4 and treated at low temperature of 4 ℃ for 6 days, so that the re-adsorption capacity of the purification columns to the same plasmid is improved, but the pollution condition of nucleic acid residues to other nucleic acid extraction is not considered, and the treatment method needs low temperature condition and takes long time. Chinese patent No. 201110294478.6 discloses a silica membrane centrifugal adsorption column regeneration liquid and its application method, the treatment method has mild condition and is ecological and environment-friendly, but the regeneration liquid has complex components and complex operation, and is not suitable for large-scale treatment and regeneration of the nucleic acid purification column.
Disclosure of Invention
The invention provides a regeneration method of a silicon matrix nucleic acid purification column, which degrades and removes residual nucleic acid on a silicon matrix membrane on the purification column and the inner wall of a tube thereof through the combined action of food-grade citric acid and a mild detergent Trition X-100, and then treats the degraded and removed residual nucleic acid with low-concentration Tris-HCl buffer solution to achieve the purposes of eliminating the residual nucleic acid and recovering the adsorption capacity of the column. The method has the advantages of mild treatment conditions, simple operation, no need of strong acid and strong alkali reagents, ecological environmental protection and the like, and can treat and regenerate a large amount of nucleic acid purification columns at one time. The purification column regenerated 5 times has nucleic acid adsorbing rate maintained at 95% and no influence on subsequent relevant experiment. The method is suitable for the silicon substrate nucleic acid purification column commonly used in the current market, the regenerated purification column comprises a silicon substrate DNA purification column and a silicon substrate RNA purification column, and the DNA purification column and the RNA purification column are separately treated only when the column is regenerated.
The technical scheme for solving the technical problems is as follows:
the solution A and the solution B required by the regeneration method of the nucleic acid purification column are prepared by deionized water, the solution A is a mixed solution of 2-4% (W/V) citric acid (food grade) and 0.025-0.1% (V/V) Trition X-100, and the solution B is a 5-10 mM Tris-HCl (pH 8.0) buffer solution.
The regeneration method of the silicon substrate nucleic acid purification column of the invention is characterized in that the solution A is preferably a mixed solution containing 2% (W/V) citric acid (food grade) and 0.025% (V/V) Trition X-100, and the solution B is preferably 5mM Tris-HCl (pH 8.0) buffer solution.
The invention can regenerate a plurality of silicon substrate nucleic acid purification columns at the same time, and the steps are as follows:
separating a batch of used purification columns from an external collection pipe, soaking the purification columns in 2 times of the volume of the solution A, slightly stirring for 10-15 hours at room temperature under the condition of 20r/min, and then pouring the used solution A; washing the purification column with tap water for 2 times; soaking the purification column in 2 times of the volume of the solution B, stirring uniformly, and standing to keep the pH of the solution B at a neutral level; taking out the purification column, soaking in 1 time volume of purified water, and sterilizing at 121 deg.C for 15 min; fishing out the purification column, and drying in a 60 ℃ oven for later use; and (4) washing the corresponding collecting pipe with tap water, and airing.
Compared with the prior art, the invention has the advantages of substantive characteristics and obvious advantages:
the invention comprehensively analyzes the problems and the defects of the existing various silicon substrate nucleic acid centrifugal purification column regeneration methods, pertinently adopts an effective method, overcomes the problems of strong acid, strong alkali, high-temperature heating treatment and the like used for cleaning and regenerating the residual nucleic acid of the existing silicon substrate nucleic acid purification column, properly prolongs the treatment time by using the dual functions of citric acid (food grade) and mild detergent Trition X-100, comprehensively degrades and removes the residual nucleic acid and other impurities on the silicon substrate membrane and the inner wall of the tube of the purification column, and effectively recovers the binding capacity of the purification column to the nucleic acid by the balance function of Tris-HCl buffer solution. The medicine used in the invention is a conventional reagent in a laboratory, and the citric acid used in the invention is food safety grade, is a large package, has low cost and is non-toxic and harmless. The method of the invention can effectively remove the residual biomolecules such as nucleic acid, protein impurities and the like on the silicon substrate purification column, and recover the nucleic acid binding capacity of the used silicon substrate purification column, so that the method can be applied to the extraction and purification of other nucleic acid samples again, and the cross contamination among the samples can not be caused. The method has the advantages of low cost, economy, high efficiency and small environmental pollution, can treat a large amount of silicon substrate nucleic acid purification columns at one time, recycles the purification columns for at least 5 times, and greatly saves the cost of experimental consumables.
Drawings
FIG. 1 is a diagram of agarose gel electrophoresis analysis of PCR products purified by the novel nucleic acid purification cartridge and the regenerated purification cartridge
FIG. 2 agarose gel electrophoresis analysis of recombinant plasmid extracted from new nucleic acid purification column and regenerated purification column and its restriction enzyme products
FIG. 3 agarose gel electrophoresis analysis of PCR product detection of corresponding eluents of regenerated or unreduced nucleic acid purification columns
Detailed Description
The present invention is further described with reference to the following examples, which should not be construed as limiting the scope of the invention.
Example 1
Adding 40g of citric acid (anhydrous citric acid) into 2000mL of deionized water, fully stirring until the citric acid is completely dissolved, adding 0.5mL of Trition X-100, and fully stirring to prepare Trition X-100 solution A containing 2% (W/V) of citric acid and 0.025% (V/V); 1.58g of Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride) was weighed and added to 2000mL of deionized water, and the mixture was sufficiently stirred until completely dissolved, and the pH was adjusted to 8.0, to obtain 5mM Tris-HCl (pH 8.0) buffer as a solution B.
The method of the invention is used for processing and regenerating the silica matrix nucleic acid purification column, and the operation steps are as follows:
the mark on the used nucleic acid purification column is wiped clean by 75% alcohol (or absolute ethyl alcohol), the purification column is separated from an external collection pipe, the purification column is soaked in 2 times of volume of solution A, the solution A is slightly stirred for 10-15 hours at the room temperature under the condition of 20r/min, the treated solution A is poured out, and the purification column is washed by tap water for 2 times. Soaking the purification column in 2 times of the solution B overnight, and detecting with pH paper until the pH value of the solution is 7.0. Taking out the purification column, soaking in a triangular flask containing 1 volume of purified water, and sterilizing at 121 deg.C for 15 min; taking out the purification column, oven drying at 60 deg.C to obtain sterile pollution-free purification column, washing with tap water, and air drying.
Example 2
Adding 60g of citric acid (anhydrous citric acid) into 2000mL of deionized water, fully stirring until the citric acid is completely dissolved, adding 1.0mL of Trition X-100, and fully stirring to prepare a Trition X-100 solution A containing 3% (W/V) of citric acid and 0.05% (V/V); 3.16g of Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride) was weighed and added to 2000mL of deionized water, and the mixture was sufficiently stirred until completely dissolved, and the pH was adjusted to 8.0, whereby 10mM Tris-HCl (pH 8.0) buffer, that is, solution B, was obtained.
Solution A and solution B were prepared according to the present example, and the used silica matrix nucleic acid purification column was subjected to the regeneration treatment according to the procedure of example 1.
Example 3
Adding 80g of citric acid (anhydrous citric acid) into 2000mL of deionized water, fully stirring until the citric acid is completely dissolved, adding 2.0mL of Trition X-100, and fully stirring to prepare Trition X-100 solution A containing 4% (W/V) of citric acid and 0.1% (V/V); 3.16g of Tris-HCl (Tris-hydroxymethyl-aminomethane hydrochloride) was weighed and added to 2000mL of deionized water, and the mixture was sufficiently stirred until completely dissolved, and the pH was adjusted to 8.0, whereby 10mM Tris-HCl (pH 8.0) buffer, that is, solution B, was obtained.
Solution A and solution B were prepared according to the present example, and the used silica matrix nucleic acid purification column was subjected to the regeneration treatment according to the procedure of example 1.
To better describe the method of the present invention, the present inventors have used a new purification column and a purification column regenerated 5 times to clone an escherichia coli putative oxidoreductase (reactive oxidoreductase) gene yqhD as an experimental object, and have performed experiments such as PCR product purification, recombinant plasmid extraction and enzyme digestion, PCR verification of purification column eluate, plasmid transformation, and the like, respectively, to verify the treatment and regeneration effects of the method of the present invention on a silicon substrate nucleic acid purification column.
The new purification column refers to an unused column attached to a commercial kit, and the regenerated purification column (used, column obtained by the regeneration treatment of the present invention) and the new purification column are the same product of the same supplier and the same batch.
Because the stability of RNA is extremely poor and the RNA is easy to degrade outside cells, the invention takes genome DNA and plasmid DNA which have better stability than the RNA as research objects, and researches the degradation and elimination effect of the method on the DNA, thereby determining the regeneration method of the silica matrix nucleic acid purification column, which is suitable for the silica matrix DNA purification column and the silica matrix RNA purification column.
The experimental materials included: escherichia coli genome extraction kit, PCR product purification kit and plasmid extraction kit, etc. are all products of domestic brand companies, and nucleic acid purification columns attached to each kit are silicon substrate nucleic acid purification columns. Other experimental materials: escherichia coli JM109, Escherichia coli K-12, cloning plasmid pGEM-3zf (+), related DNA polymerase, endonuclease, ligase and the like. M1: W2003 DNA Marker, M2: lambda DNA/HindIII DNA Marker, PCR instrument Biometra UNOII Thermoblock. The extraction of Escherichia coli genome, the purification of PCR product, the extraction of plasmid DNA, the restriction enzyme digestion and connection, the preparation and transformation of competent cells, the screening of LB culture medium, ampicillin and recon, the electrophoresis analysis, the culture of recombinant bacteria and other related operations are carried out according to related instructions and related methods.
Escherichia coli (Escherichia coli) K-12 (NC-000913) yqhD gene sequence annotated in GenBank is 1164bp, and a primer P1 (5' -TA) is designed according to the complete nucleotide sequenceGAATTCCATGGACAACTTTAATCTGCAC-3 ') and primer P2 (5'-GTAGAATTC CGAAAACGAAAGTTTGAGGC-3'), primer P1 plus EcoRI and NcoI cleavage sites, primer P2 plus EcoRI cleavage sites for convenience of gene cloning and expression in subsequent experiments. Coli k-12 genome DNA is used as a template, and a PCR technology is utilized to amplify the putative oxidoreductase gene yqhD, wherein the PCR reaction system and the amplification conditions are as follows: 50 μ L of PCR reaction: 5 μ L of 5 XPS PCR buffer,dNTP 200 u mol/L, primer each 10pmol/L, Escherichia coli K-12 genome template 50 ~ 100ng, PrimeSTAR HS DNA polymerase 1.0 unit, adding ultrapure water to the reaction total volume of 50.0 u L. The PCR amplification procedure was as follows: 3min at 95 ℃; 30sec at 95 ℃; 30sec at 58 ℃; the extension temperature and the extension time are 72 ℃ for 1.5 min; circulating for 35 times; after further extension at 72 ℃ for 10min, the reaction was terminated, 5. mu.L of the PCR product was analyzed by electrophoresis on 1.0% agarose gel to confirm that the PCR reaction was successful, and it was purified by a purification column.
The reagents and operation method used for PCR product purification are all carried out according to the kit instructions, 5 times of purification columns (the same batch and unused new purification columns are used as controls) are recycled by the invention, the same amount of PCR products are taken for purification and electrophoretic detection analysis, and the electrophoretic effect of the purified PCR products is shown in figure 1. M1 in FIG. 1 is W2003 DNA Marker (Welshi et al, Biotechnology, 2004, 14(5)), and 33-35. all Lane 1, 2, 3 and 4 in FIG. 1 have the occurrence of a band of nucleic acid of about 1.2kb in size, and the yqhD gene is identical in size, wherein Lane 1, 2 and 3 are analytical graphs of PCR products purified by 5 regenerations of purification columns of examples 1, 2 and 3 of the present invention, respectively, and Lane 4 is an analytical graph of PCR products purified by a new purification column of the same batch, which has not been used. It can be seen that the nucleic acid purification columns regenerated 5 times by the methods of examples 1, 2 and 3 have nucleic acid adsorption capacity almost equivalent to that of the new purification column, and have nucleic acid adsorption rate of 95% or more, wherein the purification column regenerated by the method of example 1 has even better nucleic acid purification effect (Lane 1) than the new purification column (Lane 4) and has improved adsorption capacity, indicating that the regeneration method of the present invention can effectively treat such purification columns, so that such purification columns can be regenerated and reused, thereby reducing the cost of experimental consumables.
The present inventors mixed the PCR products purified by the regeneration purification columns of examples 1, 2 and 3, and performed blunt end ligation with the cloning vector pGEM-3zf (+) to obtain recombinant plasmid pGEM-yqhD, which was transformed into competent cells of escherichia coli host bacterium e.coli JM109, and finally obtained recombinant strain e.coli JM109/pGEM-yqhD after screening and enzyme digestion verification, indicating that the purified nucleic acids of the regenerated purification columns do not affect the ligation reaction and transformation effect between nucleic acid fragments.
In order to verify the yield of the extracted plasmid and the restriction enzyme effect of the plasmid by the regenerated purification column, the reagent and the operation method used by the inventor for extracting the recombinant plasmid are carried out according to the instruction of a kit, the recombinant plasmid pGEM-yqhD is extracted from the recombinant strain E.coli JM109/pGEM-yqhD by regenerating the purification column for 5 times (taking a new purification column which is not used in the same batch as a reference), the equivalent recombinant plasmid is cut by EcoRI, the equivalent cut product is analyzed by agarose gel electrophoresis, and as shown in Lane 1, 2, 3 and 5 of figure 2, fragments with the cut products of about 3.2kb and 1.2kb are obtained, and the sizes of the fragments respectively correspond to the sizes of the cloning vectors pGEM-3zf (+) and yqhD. In FIG. 2, M1 is W2003 DNA Marker, M2 is lambda DNA/HindIIIDNA Marker, Lane 1, Lane 2 and Lane 3 are the cleavage results of recombinant plasmid extracted from the purification column regenerated 5 times by the methods of examples 1, 2 and 3 of the present invention, respectively, and Lane 4 (plasmid extracted from new purification column, not cleaved) and Lane5 are the cleavage results of recombinant plasmid extracted from new purification column. As can be seen from FIG. 2, the electrophoresis brightnesses of the cleavage products of pGEM-yqhD recombinant plasmids shown by Lane 1, Lane 2, Lane 3 and Lane5 are almost the same, which indicates that the nucleic acid adsorption capacity of the nucleic acid purification column regenerated 5 times by the method of the present invention is not much different from that of the new purification column when the recombinant plasmid DNA is extracted, the nucleic acid adsorption rate is maintained at 95% or more, and the cleavage effect of the extracted recombinant plasmid is not affected.
In order to detect the residual condition of nucleic acid on the silicon substrate membrane of the purification column regenerated by the present invention and to examine the influence of nucleic acid extracted from the regenerated purification column on the PCR reaction, etc., the inventors used the regenerated purification column to extract DNA of recombinant plasmid pGEM-yqhD and Escherichia coli K-12 genome, respectively, regenerated these purification columns by the methods of the above examples 1, 2 and 3, eluted these purification columns with 50. mu.L of sterile ultrapure water, respectively, and performed the plasmid transformation experiment (transformed into competent cells of Escherichia coli host bacterium E.coli JM 109) and the PCR detection experiment by the corresponding methods, respectively, on the corresponding eluents. For the purification column for extracting the recombinant plasmid pGEM-yqhD, the experimental result shows that no bacterial colony appears on an LB culture medium plate after the eluent of the regenerated purification column is transformed; under the same conditions, colonies appear on an LB medium plate after the eluent of the non-regenerated purification column is transformed (after the colonies are cultured, plasmid extraction, enzyme digestion verification and electrophoresis analysis are carried out, and the result is the same as that of the result shown in figure 2), which shows that the method can effectively remove the plasmid DNA remained on the silicon substrate purification column, and the plasmid DNA remained on the silicon substrate membrane of the regenerated purification column is not detected. The inventors carried out PCR reaction and agarose gel electrophoresis analysis by the above-described yqhD gene amplification method using each of the eluents as a template, and the results are shown in FIG. 3. M1 in FIG. 3 is W2003 DNA Marker, Lane 1 is the PCR detection result with the eluent of the purification column (without regeneration treatment) as the template after the Escherichia coli K-12 genome is extracted; lane 2 is the PCR detection result with the eluent of the purification column (without regeneration treatment) as the template after extracting the plasmid; lane 3, 4 and 5 in FIG. 3 are the PCR detection results of the eluents of the purification columns regenerated by the methods of examples 1, 2 and 3 respectively after the recombinant plasmids are extracted as templates; lane 6 is the PCR detection result of the column regenerated after the Escherichia coli K-12 genome is extracted and the eluent is the template. The research result shows that whether genome DNA or plasmid DNA is extracted, a small amount of nucleic acid remains on the corresponding nucleic acid purification column, the PCR detection result is positive, and a yqhD gene band appears, and the nucleic acid extracted by the regeneration purification columns is taken as a template and does not influence the corresponding PCR reaction (Lane 1 and Lane 2 in FIG. 3). The inventors have confirmed that the regeneration treatment of such a nucleic acid purification column by the method of the present invention can effectively remove the residual nucleic acid components on the silicon substrate membrane and the inner wall of the tube thereof, and that the PCR detection result is negative and no target band of the yqhD gene appears ( Lane 3, 4, 5 and 6 in FIG. 3). In conclusion, the regeneration method of the invention can effectively remove the biomolecules such as residual nucleic acid, protein impurities and the like on the purification column, and recover the nucleic acid binding capacity of the used silica matrix nucleic acid purification column, so that the regeneration method can be applied to the extraction and purification of other nucleic acid samples again, and can not cause cross contamination among samples.
The invention comprehensively analyzes some problems existing in the current regeneration treatment method of the nucleic acid purification column, and creatively optimizes and combines the latest reagent formula and treatment method. The method can effectively remove the residual nucleic acid, protein impurities and other biomolecules on the silicon substrate nucleic acid purification column and recover the nucleic acid binding capacity of the purification column. The method of the invention has the advantages of economy, high efficiency, small environmental pollution and the like, and can process a large amount of the silica matrix nucleic acid purification columns at one time. The method can regenerate and utilize the used silica matrix nucleic acid purification column for at least 5 times, thereby greatly reducing the experiment cost. The regeneration method of the silicon substrate nucleic acid purification column is also suitable for the silicon substrate DNA purification column and the silicon substrate RNA purification column which are commonly used and commercialized at present, and has applicability and universality.
It should be understood that modifications or changes may be made by those skilled in the art in light of the above teachings, such as, for example, by modifying the proportions and amounts of the reagents involved in the various solutions described above, modifying, changing and optimizing the steps involved, and all such modifications and changes are intended to fall within the scope of the invention as defined in the appended claims.
Claims (4)
1. A regeneration method of a silica matrix nucleic acid purification column is characterized in that: the purification column is treated by solution A and solution B, wherein the solution A is a mixed solution containing 2-4% (W/V) citric acid and 0.05-0.1% (V/V) Trition X-100, and the solution B is a 5-10 mM Tris-HCl (pH 8.0) buffer solution.
2. The solution a and solution B according to claim 1, characterized in that: the solution A preferably contains 2% (W/V) citric acid and 0.025% (V/V) Trition X-100, and the solution B preferably contains 5mM Tris-HCl (pH 8.0) buffer.
3. The solution according to claim 1 and claim 2 for regenerating the purification column, characterized in that it is carried out as follows: a. separating the used purification column from an external collection pipe, soaking the purification column in the solution A, slightly stirring for 10-15 hours at room temperature, and washing the purification column with tap water for 2 times; b. soaking the purification column in the solution B to balance to neutrality, then soaking the purification column in purified water to sterilize at 121 ℃ for 15 minutes, and drying the purification column in a 60 ℃ oven for later use; c. and (5) washing the collecting pipe with tap water, and airing.
4. The solution and process steps according to claim 1, claim 2 and claim 3, the regenerated silica-based nucleic acid purification column comprises silica-based DNA purification column and silica-based RNA purification column.
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09246227A (en) * | 1996-03-12 | 1997-09-19 | Japan Organo Co Ltd | Silicon wafer cleaning equipment |
| US20050032105A1 (en) * | 2001-10-12 | 2005-02-10 | Bair Robert Jackson | Compositions and methods for using a solid support to purify DNA |
| CN101717421A (en) * | 2009-12-15 | 2010-06-02 | 大连水产学院 | Method for regenerating nucleic acid purification column |
| CN101892222A (en) * | 2010-06-30 | 2010-11-24 | 清华大学 | Reusable plasmid extraction kit |
| CN102220308A (en) * | 2010-04-19 | 2011-10-19 | 韩寒 | Method for recovering plasmid extraction and purification columns |
| US20130341213A1 (en) * | 2012-06-20 | 2013-12-26 | Jolanta Vitkuté | Method to prevent silica-based column aging |
| CN105916985A (en) * | 2013-09-19 | 2016-08-31 | 诺维信公司 | Polypeptides having mannanase activity and polynucleotides encoding same |
| CN110257245A (en) * | 2019-07-16 | 2019-09-20 | 东莞博识生物科技有限公司 | Nucleic acid detection reagent card |
| CN111793978A (en) * | 2020-06-11 | 2020-10-20 | 江苏益飞环保科技有限公司 | Facing material based on plant antibacterial agent and preparation method thereof |
| US20210008552A1 (en) * | 2017-06-15 | 2021-01-14 | Lexagene, Inc. | Nucleic acid extraction and purification cartridges |
| CN116355724A (en) * | 2022-12-14 | 2023-06-30 | 厦门为正生物科技股份有限公司 | Nucleic acid extraction device and nucleic acid extraction method |
| CN116622747A (en) * | 2023-05-31 | 2023-08-22 | 广西产研院生物制造技术研究所有限公司 | Gene for coding dextran sucrase and application thereof |
-
2021
- 2021-06-03 CN CN202110616976.1A patent/CN113278609B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09246227A (en) * | 1996-03-12 | 1997-09-19 | Japan Organo Co Ltd | Silicon wafer cleaning equipment |
| US20050032105A1 (en) * | 2001-10-12 | 2005-02-10 | Bair Robert Jackson | Compositions and methods for using a solid support to purify DNA |
| CN101717421A (en) * | 2009-12-15 | 2010-06-02 | 大连水产学院 | Method for regenerating nucleic acid purification column |
| CN102220308A (en) * | 2010-04-19 | 2011-10-19 | 韩寒 | Method for recovering plasmid extraction and purification columns |
| CN101892222A (en) * | 2010-06-30 | 2010-11-24 | 清华大学 | Reusable plasmid extraction kit |
| US20130341213A1 (en) * | 2012-06-20 | 2013-12-26 | Jolanta Vitkuté | Method to prevent silica-based column aging |
| CN105916985A (en) * | 2013-09-19 | 2016-08-31 | 诺维信公司 | Polypeptides having mannanase activity and polynucleotides encoding same |
| US20210008552A1 (en) * | 2017-06-15 | 2021-01-14 | Lexagene, Inc. | Nucleic acid extraction and purification cartridges |
| CN110257245A (en) * | 2019-07-16 | 2019-09-20 | 东莞博识生物科技有限公司 | Nucleic acid detection reagent card |
| CN111793978A (en) * | 2020-06-11 | 2020-10-20 | 江苏益飞环保科技有限公司 | Facing material based on plant antibacterial agent and preparation method thereof |
| CN116355724A (en) * | 2022-12-14 | 2023-06-30 | 厦门为正生物科技股份有限公司 | Nucleic acid extraction device and nucleic acid extraction method |
| CN116622747A (en) * | 2023-05-31 | 2023-08-22 | 广西产研院生物制造技术研究所有限公司 | Gene for coding dextran sucrase and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| TATIANA ALVAREZ等: "Assessment of Different Regeneration and Washing Solutions in an Affinity Chromatography Procedure to Purify Plantibodies", CHROMATOGRAPHIA, pages 403 * |
| YI-MING TAO等: "Purification and Characterization of Polyphenol Oxidase fromJackfruit (Artocarpusheterophyllus) Bulbs", J AGRIC FOOD CHEM, vol. 61, no. 51, pages 12662 - 12669 * |
| 熊明华等: "硅基质膜核酸(DNA)纯化柱的去污染与再生", 基因组学与应用生物学, vol. 34, no. 7, pages 1468 - 1472 * |
| 董敏等: "质粒纯化利用离子交换柱的再生方法", 基础医学与临床, pages 561 - 563 * |
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